Conformational change in individual enzyme molecules
| Autor: | Frannie Itzkow, Joanna MacLean, Jeremie J. Crawford, Douglas B. Craig |
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| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Conformational change Protein Folding Hot Temperature Protein Conformation Population Electrophoretic Mobility Shift Assay Biochemistry Capillary electrophoresis Heat shock protein Enzyme Stability Escherichia coli Electrophoretic mobility shift assay Heat shock education Molecular Biology Incubation Heat-Shock Proteins Fluorescent Dyes education.field_of_study Stochastic Processes Chromatography Chemistry Escherichia coli Proteins Electrophoresis Capillary Galactose Reproducibility of Results Galactosides Cell Biology beta-Galactosidase Recombinant Proteins Electrophoresis Spectrometry Fluorescence Lac Operon Biophysics Acridines sense organs Gene Deletion Heat-Shock Response |
| Zdroj: | Biochemistry and cell biology = Biochimie et biologie cellulaire. 93(6) |
| ISSN: | 1208-6002 |
| Popis: | Single β-galactosidase molecule assays were performed using a capillary electrophoresis based protocol, employing post-column laser-induced fluorescence detection. In a first set of experiments, the distribution of single β-galactosidase molecule catalytic rates and electrophoretic mobilities were determined from lysates of Escherichia coli strains containing deletions for different heat shock proteins and grown under normal and heat shock conditions. There was no clear observed pattern of effect of heat shock protein expression on these distributions. In a second set of experiments, individual enzyme molecule catalytic rates were determined at 21 °C before and after 2 sequential brief periods of incubation at 50, 28, and 10 °C. The brief incubations at 50 °C caused a change in the enzyme molecules resulting in a different catalytic rate. Any given molecule was just as likely to show an increase in rate as a decrease, resulting in no significant difference in the average rate of the population. The average change in individual molecule rate was dependent upon the temperature of the brief incubation period, with a lesser average change occurring at 28 °C and negligible change at 10 °C. A third set of experiments was similar to that of the second with the exception that it was electrophoretic mobility that was considered. This provided a similar result. Incubation at higher temperature resulted in a change in electrophoretic mobility. The probability of an individual molecules switching to a higher mobility was approximately equal to that of switching to a lower mobility, resulting in no net average change in the population. The magnitude of the changes in electrophoretic mobilities suggest that the associated conformational changes are subtle. |
| Databáze: | OpenAIRE |
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